The present invention relates to an affinity chromatography apparatus and method.
The present invention further relates to a semi-automated apparatus and method for performing immunoassays.
The present invention more particularly relates to an apparatus and method for semi-automatically carrying out enzyme-linked immunological sandwich assays (ELISA), by means of affinity chromatography.
The present invention further relates to an apparatus and method for carrying out "sandwich-type" immunoassays by affinity paper, or other flexible support matrix chromatography, in a semi-automated fashion.
The recent biotechnological development and commercialization of monoclonal antibodies as a source of inexpensive, homogenous immunoglobulins has led to the widespread use of inexpensive, relatively easy to use, immunoassays for a wide variety of antigenic substances found in human and animal body fluids.
In sandwich assays, a sample, which may or may not contain an antigenic subject molecule X, is allowed to react with an anti-X antibody, which anti-X antibody has been previously, either covalently or otherwise, bound to a paper strip or other support matrix. Any subject molecule X in the sample which reacts with the anti-X antibody consequently becomes irreversibly bound to the paper strip or other support matrix.
Next, a conjugate, which is made of the same or another type of anti-X antibody, previously covalently bound to a marker, is allowed to react with the original subject molecule X-anti-X antibody immobilized complex. Any excess free conjugate is then washed away. In this technique, the amount of marker that becomes irreversibly bound to the complex is proportional to the amount of subject molecule X in the sample in question.
The conjugate-bound marker may be either a radioactive isotope, or it may also be an enzyme. A radioactive isotope marker may be readily measured by conventional scintillation counting. A conjugate-bound enzyme marker may be measured by whichever is the preferred way by which the particular enzyme of choice is usually measured. One of the most convenient types of conjugate-bound enzymes to use as a marker for immunoassays is that of colorimetric enzymes, or enzymes that convert a colorless, or near colorless, chromatogen into a strongly-colored, and therefore easily measurable, chromatophore. Additionally, in certain methods, fluorometric markers are also envisioned for use with the apparatus and method of the present invention. Colorimetric enzyme markers are generally easier, safer, and less expensive to use in ELISA than are radioactive markers.
The aforementioned support matrix may be preferably a flexible non-paper porous or non-porous membrane, or a flexible paper strip. In either case, ELISA constitutes a particular type of affinity chromatography.
The above comments are supplied here to provide both a background and rationale for using a colorimetric ELISA affinity chromatography method for the detection of various antigenic protein molecules or non-protein molecules of interest in human or animal body fluids, utilizing the apparatus and method of the present invention.
One of the main problems encountered in utilizing ELISA with the prior art capillary affinity chromatography systems, such as those disclosed in U.S. Pat. No. 4,094,647 by Deutsch and Mead and in U.S. Patent No. 4,690,907, Hibino and Hirato is that ELISA cannot be carried out in such systems in a single semi-automated procedure of the type to which the present invention relates. That is, after a conjugate has reacted in the above prior art systems, and the excess conjugate is washed away, the support matrix must then be physically taken out of the wash solution reservoir, and manually placed into a separate color development solution reservoir. Thus, at least two distinct manual steps or procedures are required in order to perform ELISA in prior art affinity chromatography systems. If these two manual procedures are not carefully and cleanly carried out, or not accurately timed and separated, then false positive and other spurious test results can occur, due in part to the reaction of the color development solution with excess unbound conjugate.